Low dose combination therapy for treatment of myeloproliferative neoplasms

ABSTRACT

The present application relates to treatment of myeloproliferative neoplasms using the JAK1/JAK2 inhibitor, ruxolitinib, in combination with a low dose of a Pim inhibitor, N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide, wherein the combination is unexpectedly synergistic at a very low dose of the Pim inhibitor.

TECHNICAL FIELD

The present application relates to treatment of myeloproliferativeneoplasms using the JAK1/JAK2 inhibitor, ruxolitinib, in combinationwith a low dose of a Pim inhibitor,N-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,wherein the combination is unexpectedly synergistic at a very low doseof the Pim inhibitor.

BACKGROUND

The discovery of the activating V617F mutation in Janus kinase 2 (JAK2)in the majority of patients with the classic Philadelphiachromosome-negative (Ph−) myeloproliferative neoplasms (MPN) provided astrong impetus for the development of pharmacologic inhibitors of JAK2,culminating in the regulatory approval of the JAK1/2 inhibitor,ruxolitinib, for patients with myelofibrosis (MF) in 2011 andhydroxyurea (HU)-resistant/intolerant polycythemia vera (PV) in 2014.The activity of ruxolitinib in patients with MF without regard to JAK2mutation status was an important observation that was vindicated by thefinding of universal activation of the JAK-signal transducer andactivator of transcription (STAT) pathway in MPNs.

However, because JAK2 signaling is not suppressed long term andmolecular remission is not observed in patients treated with JAK2inhibitors, there is a need for new therapies to improve patientoutcome. This application is directed to this need and others.

SUMMARY

The present application provides, inter alia, methods of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patientN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and ruxolitinib, or apharmaceutically acceptable salt thereof.

The present application further provides methods of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patient a dose from about 5 mg/day to about 100mg/dayN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of ruxolitinib, or a pharmaceutically acceptable saltthereof.

The present application also provides methods of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patient a dose from about 5 mg/day to about 100mg/dayN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and a dose of from about10 mg/day to about 50 mg/day of ruxolitinib, or a pharmaceuticallyacceptable salt thereof.

The present application further provides the use of the compounds at thedoses recited herein for use in the manufacture of medicaments for usein treating a myeloproliferative neoplasm.

The present application also provides the compounds at the doses recitedherein for use in treating a myeloproliferative neoplasm.

The details of one or more embodiments are set forth in the descriptionbelow. Other features, objects, and advantages will be apparent from thedescription and from the claims.

DESCRIPTION OF THE FIGURES

FIGS. 1(A, B, and C) depict the % growth in BaF3/EpoR/JAK2-V617F, UKE1,and SET2 cells, respectively, for untreated cells or cells treated withruxolitinib (Rux) or Compound 1 (PIMi), and these two together(Rux+PIMi).

FIGS. 2 (A and B) depict % annexin V+ in UKE1 and SET2 cells treatedwith vehicle (DMSO), ruxolitinib, Compound 1 (PIMi), or the two drugstogether.

FIG. 3 depicts the % growth in peripheral blood mononuclear cells froman MPN patient (PV, JAK2-V617F+) in the presence of either DMSO-vehicle,50 nM ruxolitinib (Rux), 20 nM or 5 nM Compound 1 (PIMi) or the twodrugs in combination. Epo-independent erythroid colonies were scoredafter 12 days. Results are shown in FIG. 3. Expected % growth ofcombinations using the Bliss independence model is indicated by thetransparent bars with dotted lines. The lower measured % growthindicates synergy.

FIG. 4 depicts immunoblot analysis of MPN patient granulocytes leftuntreated (DMSO only) or treated with the indicated concentrations ofCompound 1 (PIMi) or ruxolitinib.

FIG. 5(A) depicts % pBAD inhibition in plasma samples from patients at24 hours post dose of Compound 1.

FIG. 5(B) depicts % pBAD inhibition in plasma samples from patients aton cycle 1, day 8 for 80 mg BID of Compound 1.

DETAILED DESCRIPTION

For the terms “e.g.” and “such as,” and grammatical equivalents thereof,the phrase “and without limitation” is understood to follow unlessexplicitly stated otherwise.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, the term “about” means “approximately” (e.g., plus orminus approximately 10% of the indicated value).

The present invention relates to the use of a JAK1/JAK2 inhibitor,ruxolitinib, in combination with a PIM inhibitor for treatment of amyeloproliferative neoplasm. It has been unexpectedly found that verylow doses of the PIM inhibitor synergize with ruxolitinib at levelsbelow IC₅₀ and much lower for the PIM inhibitor.

Ruxolitinib,(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,is an inhibitor of JAK1 and JAK2. The IC₅₀ of ruxolitinib was measuredby Assay A infra at 1 mM ATP and found to be less than 10 nM at JAK1 andJAK2.

Ruxolitinib can be made by the procedure described in U.S. Pat. No.7,598,257 (Example 67), filed Dec. 12, 2006, which is incorporatedherein by reference in its entirety. Ruxolitinib phosphate can beprepared as described in US 2008/0312259, which is incorporated hereinby reference in its entirety.

N-{(7R)-4-[(3R,4R,5S)-3-Amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide(Compound 1 below) is an inhibitor of Pim1, Pim2, and Pim3. Compound 1can prepared as described in U.S. Pat. No. 9,200,004 or US 2017/0121310,each of which is incorporated herein by reference in its entirety. TheIC₅₀ of Compound 1 was measured by the procedure in Assay B infra andfound to be ≤100 nM at Pim1, Pim2, and Pim3. The phosphoric acid salt,dihydrochlorlic acid salt, hydrochloric acid salt, maleic acid salt,adipic acid salt, hydrobromic acid salt, (R)-(−)-mandelic acid salt,salicylic acid salt, benzoic acid salt, benzenesulfonic acid salt,L-pyroglutamic acid salt, methanesulfonic acid salt,(1S)-(+)-10-camphorsulfonic acid salt, fumaric acid salt, sulfuric acidsalt, L-tartaric acid salt, and D-tartaric acid salt of Compound 1 canbe prepared as described in US 2017/0121310, which is incorporatedherein by reference in its entirety. The IC₅₀ of the dihydrochloric acidand phosphoric acid salts of Compound 1 was measured by the procedure inAssay B infra and found to be <35 nM at Pim1, Pim2, and Pim3.

Accordingly, the present application provides a method of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patientN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and ruxolitinib, or apharmaceutically acceptable salt thereof.

The present application further provides a method of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patient a dose from about 2 mg/day to about 160mg/dayN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of ruxolitinib, or a pharmaceutically acceptable saltthereof.

The present application further provides a method of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patient a dose from about 2 mg/day to about 100mg/dayN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of ruxolitinib, or a pharmaceutically acceptable saltthereof.

The present application further provides a method of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patient a dose from about 5 mg/day to about 100mg/dayN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of ruxolitinib, or a pharmaceutically acceptable saltthereof.

The present application also provides a method of treating amyeloproliferative neoplasm in a patient in need thereof, comprisingadministering to said patient a dose from about 5 mg/day to about 100mg/dayN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and a dose of from about10 mg/day to about 50 mg/day of ruxolitinib, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 5 mg/day toabout 100 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 80 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 70 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg toabout 60 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 50 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 40 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 30 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 20 mg/day.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 2 mg BID toabout 50 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 40 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 35 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg toabout 30 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 25 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 20 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 15 mg BID.

In some embodiments, the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 10 mg BID.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 10 mg/day to about 30 mg/day.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 10 mg/day to about 50 mg/day.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 10 mg/day to about 30 mg/day.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 10 mg/day.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about about 20 mg/day.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 30 mg/day.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 5 mg BID to about 25 mg BID.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 5 mg BID to about 15 mg BID.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 5 mg BID.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about about 10 mg BID.

In some embodiments, the dose of ruxolitinib, or a pharmaceuticallyacceptable salt thereof, is about 15 mg BID.

In some embodiments, ruxolitinib, or a pharmaceutically acceptable saltthereof, is ruxolitinib phosphate.

In some embodiments, theN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is selected from thephosphoric acid salt, dihydrochlorlic acid salt, hydrochloric acid salt,maleic acid salt, adipic acid salt, hydrobromic acid salt,(R)-(−)-mandelic acid salt, salicylic acid salt, benzoic acid salt,benzenesulfonic acid salt, L-pyroglutamic acid salt, methanesulfonicacid salt, (1S)-(+)-10-camphorsulfonic acid salt, fumaric acid salt,sulfuric acid salt, L-tartaric acid salt, and D-tartaric acid salt ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

In some embodiments, theN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is the phosphoric acidsalt ofN-{(7R)-4-[(3R,4R,5S)-3-Amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.

In some embodiments, the myeloproliferative neoplasm is selected frompolycythemia vera (PV), essential thrombocythemia (ET), primarymyelofibrosis, chronic myelogenous leukemia (CML), chronicmyelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES),systemic mast cell disease (SMCD), chronic neutrophilic leukemia (CNL),and chronic eosinophilic leukemia.

In some embodiments, the myeloproliferative neoplasm is selected frompolycythemia vera (PV).

In some embodiments, the myeloproliferative neoplasm is selected fromessential thrombocythemia (ET).

In some embodiments, the myeloproliferative neoplasm is selected fromprimary myelofibrosis.

In some embodiments, the myeloproliferative neoplasm is myelofibrosis.

In some embodiments, the ruxolitinib, or pharmaceutically acceptablesalt thereof, and theN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, are administered orally.

The preceding embodiments are intended to be combined in any suitablecombination as if the embodiments are multiply dependent claims (e.g.,the embodiments related to the individual doses for ruxolitinib, theembodiments related to the individual doses for the PIM inhibitor(Compound 1), the embodiments related to the salt forms, the embodimentsrelated to the individual types of myeloproliferative neoplasms, and theembodiments related to oral administration can be combined in anycombination). The combinations are not separately listed herein merelyfor the sake of brevity.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.,hydrates and solvates) or can be isolated. When in the solid state, thecompounds described herein and salts thereof may occur in various formsand may, e.g., take the form of solvates, including hydrates. Thecompounds may be in any solid state form, such as a polymorph orsolvate, so unless clearly indicated otherwise, reference in thespecification to compounds and salts thereof should be understood asencompassing any solid state form of the compound.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. The term “pharmaceutically acceptablesalts” refers to derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt form. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The pharmaceutically acceptable saltsof the present invention include the non-toxic salts of the parentcompound formed, e.g., from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, alcohols (e.g., methanol, ethanol,iso-propanol, or butanol) or acetonitrile (MeCN) are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J.Pharm. Sci., 1977, 66(1), 1-19, and in Stahl et al., Handbook ofPharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). Insome embodiments, the compounds described herein include the N-oxideforms.

The dosages described herein are on a free base basis.

The terms “individual” or “patient,” used interchangeably, refer to anyanimal, including mammals, preferably mice, rats, other rodents,rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and mostpreferably humans.

The phrase “therapeutically effective amount” refers to the amount ofactive compound or pharmaceutical agent that elicits the biological ormedicinal response in a tissue, system, animal, individual or human thatis being sought by a researcher, veterinarian, medical doctor or otherclinician.

The term “treating” or “treatment” refers to one or more of (1)inhibiting the disease; e.g., inhibiting a disease, condition ordisorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,arresting further development of the pathology and/or symptomatology);and (2) ameliorating the disease; e.g., ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., reversing the pathology and/or symptomatology) such as decreasingthe severity of disease. In one embodiment, treating or treatmentincludes preventing or reducing the risk of developing the disease;e.g., preventing or reducing the risk of developing a disease, conditionor disorder in an individual who may be predisposed to the disease,condition or disorder but does not yet experience or display thepathology or symptomatology of the disease. The term “BID” means twotimes a day.

Additional Combinations

Cell growth and survival can be impacted by multiple signaling pathways.Thus, it is useful to combine different kinase inhibitors, exhibitingdifferent preferences in the kinases which they modulate the activitiesof, to treat such conditions. Targeting more than one signaling pathway(or more than one biological molecule involved in a given signalingpathway) may reduce the likelihood of drug-resistance arising in a cellpopulation, and/or reduce the toxicity of treatment.

Accordingly, the methods can further comprise the administration of oneor more other kinase inhibitors. For example, the compounds of theinvention can be combined with one or more inhibitors of the followingkinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG,PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR,HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, CSFIR, KIT,FLK-II, KDR/FLK-1, FLK-4, fit-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron,Sea, TRKA, TRKB, TRKC, FLT3, VEGFR/Flt2, Flt4, EphA1, EphA2, EphA3,EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL,ALK and B-Raf. Additionally, the Pim inhibitors of the invention can becombined with inhibitors of kinases associated with the PIK3/Akt/mTORsignaling pathway, such as PI3K, Akt (including Akt1, Akt2 and Akt3) andmTOR kinases.

The methods can further be used in combination with other methods oftreatment, for example by chemotherapy, irradiation, or surgery. Thecompounds can be administered in combination with one or moreanti-cancer drugs, such as a chemotherapeutics. Examplechemotherapeutics include any of: abarelix, aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide,asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin,bortezombi, bortezomib, busulfan intravenous, busulfan oral,calusterone, capecitabine, carboplatin, carmustine, cetuximab,chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib,daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane,docetaxel, doxorubicin, dromostanolone propionate, eculizumab,epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide,exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine,fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumabozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin,leuprolide acetate, levamisole, lomustine, meclorethamine, megestrolacetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycinC, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine,nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab,pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin,pipobroman, plicamycin, procarbazine, quinacrine, rasburicase,rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinibmaleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide,thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab,tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,vinorelbine, vorinostat and zoledronate.

The methods can further be used in combination with one or moreanti-inflammatory agents, steroids, immunosuppressants, or therapeuticanti-bodies.

When more than one pharmaceutical agent is administered to a patient,they can be administered simultaneously, sequentially, or in combination(e.g., for more than two agents).

Compositions

The compounds can be administered in the form of pharmaceuticalcompositions. These compositions can be prepared in a manner well knownin the pharmaceutical art, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is indicatedand upon the area to be treated. Administration may be topical(including transdermal, epidermal, ophthalmic and to mucous membranesincluding intranasal, vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be,e.g., by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

The pharmaceutical compositions can contain, as the active ingredient,the compounds, or a pharmaceutically acceptable salt thereof, incombination with one or more pharmaceutically acceptable carriers(excipients). In some embodiments, the composition is suitable fortopical administration. In making the compositions, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, e.g., a capsule,sachet, paper, or other container. When the excipient serves as adiluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, e.g.,up to 10% by weight of the active compound, soft and hard gelatincapsules, suppositories, sterile injectable solutions and sterilepackaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g., about 40 mesh.

The compounds may be milled using known milling procedures such as wetmilling to obtain a particle size appropriate for tablet formation andfor other formulation types. Finely divided (nanoparticulate)preparations of the compounds of the invention can be prepared byprocesses known in the art see, e.g., WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; and sweetening agents and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

In some embodiments, the pharmaceutical composition comprises silicifiedmicrocrystalline cellulose (SMCC) and at least one compound describedherein, or a pharmaceutically acceptable salt thereof. In someembodiments, the silicified microcrystalline cellulose comprises about98% microcrystalline cellulose and about 2% silicon dioxide w/w.

In some embodiments, the composition is a sustained release compositioncomprising at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier. In some embodiments, the composition comprises at least onecompound described herein, or a pharmaceutically acceptable saltthereof, and at least one component selected from microcrystallinecellulose, lactose monohydrate, hydroxypropyl methylcellulose andpolyethylene oxide. In some embodiments, the composition comprises atleast one compound described herein, or a pharmaceutically acceptablesalt thereof, and microcrystalline cellulose, lactose monohydrate andhydroxypropyl methylcellulose. In some embodiments, the compositioncomprises at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and microcrystalline cellulose, lactosemonohydrate and polyethylene oxide. In some embodiments, the compositionfurther comprises magnesium stearate or silicon dioxide. In someembodiments, the microcrystalline cellulose is Avicel PH102™. In someembodiments, the lactose monohydrate is Fast-flo 316™. In someembodiments, the hydroxypropyl methylcellulose is hydroxypropylmethylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/orhydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel K00LV™). Insome embodiments, the polyethylene oxide is polyethylene oxide WSR 1105(e.g., Polyox WSR 1105™).

The components used to formulate the pharmaceutical compositions are ofhigh purity and are substantially free of potentially harmfulcontaminants (e.g., at least National Food grade, generally at leastanalytical grade, and more typically at least pharmaceutical grade).Particularly for human consumption, the composition is preferablymanufactured or formulated under Good Manufacturing Practice standardsas defined in the applicable regulations of the U.S. Food and DrugAdministration. For example, suitable formulations may be sterile and/orsubstantially isotonic and/or in full compliance with all GoodManufacturing Practice regulations of the U.S. Food and DrugAdministration.

The active compound may be effective over a wide dosage range and isgenerally administered in a therapeutically effective amount. It will beunderstood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight and response of the individual patient, the severity of thepatient's symptoms and the like.

The therapeutic dosage of a compound of the present invention can varyaccording to, e.g., the particular use for which the treatment is made,the manner of administration of the compound, the health and conditionof the patient, and the judgment of the prescribing physician. Theproportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, e.g., about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face mask, tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, e.g., liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline®(petroleum jelly) and the like. Carrier compositions of creams can bebased on water in combination with glycerol and one or more othercomponents, e.g., glycerinemonostearate, PEG-glycerinemonostearate andcetylstearyl alcohol. Gels can be formulated using isopropyl alcohol andwater, suitably in combination with other components such as, e.g.,glycerol, hydroxyethyl cellulose and the like.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers or stabilizers will resultin the formation of pharmaceutical salts.

Kits

The present application also includes pharmaceutical kits useful, whichinclude one or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of the compound, or any ofthe embodiments thereof. Such kits can further include one or more ofvarious conventional pharmaceutical kit components, such as, e.g.,containers with one or more pharmaceutically acceptable carriers,additional containers, etc., as will be readily apparent to thoseskilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results. The compounds of the Examples have been found to bePim-kinase inhibitors according to at least one assay described herein.

EXAMPLES Example A: In Vitro JAK Kinase Assay

Compounds herein were tested for inhibitory activity of JAK targetsaccording to the following in vitro assay described in Park et al.,Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a.781-1124) were expressed using baculovirus in insect cells and purified.The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuringthe phosphorylation of a biotinylated peptide. The phosphorylatedpeptide was detected by homogenous time resolved fluorescence (HTRF).IC₅₀s of compounds were measured for each kinase in the 40 μL reactionsthat contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8)buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1mM IC₅₀ measurements, ATP concentration in the reactions was 1 mM.Reactions were carried out at room temperature for 1 hour and thenstopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assaybuffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeledantibody took place for 40 minutes and HTRF signal was measured on aPHERA star plate reader (BMG, Cary, N.C.). The data for the JAK1 and/orJAK2 inhibitors were obtained by testing the compounds in the Example Aassay at 1 mM ATP.

Example B. Pim Enzyme Assays

Pim-1 and Pim-3 kinase assays—20 μL reactions were run in white 384 wellpolystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer(50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl₂, 0.01% BSA, 5 mM DTT),containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269),1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3,Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped byaddition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mMEDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112)Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donorbeads (PerkinElmer 6760002) along with Protein-A acceptor beads(PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOPbuffer with beads and stopping the reactions were done under reducedlight. Prior to the stopping reactions STOP buffer with beads waspre-incubated for 1 h in the dark at room temperature. After stoppingthe reactions, plates were incubated for 1 h in the dark at roomtemperature before reading on a PHERAstar FS plate reader (BMG Labtech)under reduced light.

Pim-2 kinase assay—20 μL reactions were run in white 384 wellpolystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer(50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl₂, 0.01% BSA, 5 mM DTT),containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate(Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET DilutionBuffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreenTb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubationat room temperature, plates were read on a PHERAstar FS plate reader(BMG Labtech).

Compounds of the invention having an IC₅₀ of 2 μM or less when testedfor PIM kinase activity under the assay conditions disclosed above areconsidered active.

Although the above in vitro assays are conducted at 1 mM ATP compoundscan also be evaluated for potency and in vitro activity against PIMtargets utilizing K_(m) conditions, where the concentration of ATP isset to the K_(m) value and the assay is more sensitive to PIM inhibitionactivity.

Example C. Pim Cellular Assays

One or more compounds of the invention were tested for inhibitoryactivity of PIM according to at least one of the following cellularassays. Compounds of the invention having an IC₅₀ of 10 μM or less whentested for PIM kinase activity under the cellular assay conditionsdisclosed below would be and were considered active.

Pim Cell Proliferation Assays

KG-1A cells are purchased from ATCC (Manassas, Va.) and KMS.12.BM cellsare purchased from NIBIO, JCRB cell bank (Tokyo, Japan) and maintainedin the culture mediums recommended, RPMI, 10% FBS (Roswell Park MemorialInstitute 1640 Medium supplemented with 10% fetal bovine serum) and IMDM20% FBS (Iscove's Modified Dulbecco's Medium (MDM) with 20% fetal bovinestrum) (Mediatech, Manassas, Va.) respectively. To measure theanti-proliferation activity of test compounds, both cell lines areplated with the culture medium (2×10³ cells/well/in 200 μL) into 96-wellpolystyrene ultralow binding (Costar®) in the presence or absence of aconcentration range of test compounds. After 4 days, [³H]-thymidine, 1μCi/10 μL/well (PerkinElmer, Boston, Mass.) in culture medium is thenadded to the cell culture for an additional 16 h before the incorporatedradioactivity is separated by filtration with a Packard MicroplateHarvester with water through a 0.3% polyethylenimine pre-wetted glassfiber GF/B filter plates (Packard Bioscience/PerkinElmer, Boston,Mass.). The plate is measured by liquid scintillation counting with aTopCount® scintillation sounter (PerkinElmer). IC₅₀ determination isperformed by fitting the curve of percent inhibition versus the log ofthe inhibitor concentration using GraphPad Prism® 5.0 software.

Pim pBAD Signaling Assays

KG-1A cells are purchased from ATCC (Manassas, Va.) and KMS.12.BM cellsare purchased from NIBIO, JCRB cell bank (Tokyo, Japan) and maintainedin the culture mediums recommended, RPMI, 10% FBS and IMDM 20% FBS(Mediatech, Manassas, Va.) respectively. To measure the pBAD inhibitoryactivity of the compounds, both cell lines are plated with the culturemedium (1×10⁶/well/100 μL for KG1A and 4×10⁵ cells/well/in 100 μL forKMS12BM) into 96-well V bottom polypropylene plates (Matrix, ThermoFisher, USA) and incubated 30 min. at 37° C. to normalize cell signalingfrom handling. Test compounds are added at an appropriate concentrationrange and further incubated for 2.5 h for KMS.12.BM cells and 4 h forKG1-A cells. Plates are centrifuged at 2000 RPM for 10 min. andsupernatants aspirated. 100 μL lysis buffer with protease inhibitors(Cell Signaling Technologies, Danver, Mass., Sigma, St Louis Mo., EMD,USA) is added to the pellets, mixed well and set on ice for 30 min.Lysates are frozen overnight at −80° C. To measure the pBAD activity, aCell Signaling ELISA (enzyme-linked immunosorbent assay) kit (CellSignaling Path Scan phosphor pBAD ELISA) is utilized. 50 μL of thelysate is tested per the ELISA protocol and the data analysis isperformed by software on a SpectraMax® M5 plate reader (MolecularDevices, Sunnyvale, Calif.). IC₅₀ determination is performed by fittingthe curve of percent inhibition versus the log of the inhibitorconcentration using GraphPad Prism® 5.0 software.

Example 1: The Pim Inhibitor, Compound 1, Synergizes with Ruxolitinib toInhibit MPN Model Cell Growth

BaF3/EpoR/JAK2-V617F, UKE1, and SET2 cells were either left untreated(DMSO-vehicle only), or treated with ruxolitinib (Rux) or Compound 1(PIMi), and these two together (Rux+PIMi). Results forBaF3/EpoR/JAK2-V617F cells are shown in FIG. 1(A); results for UKE1cells are shown in FIG. 1(B); and results for SET2 cells are shown inFIG. 1(C). Synergy studies were performed by CellTiter-Glo at 72 hr.Expected % growth of combinations using the Bliss independence model isindicated by the dotted line: the lower measured % growth suggestssynergy.

Example 2: Compound 1 and Ruxolitinib Syngeristically Induce Apoptosis

UKE1 and SET2 cells were treated with either DMSO, ruxolitinib, Compound1 (PIMi), or these two together. Cells were stained using FITC Annexin Vand PI and analyzed by flow cytometry. Error bars indicate s.d. The pvalue was calculated by one-way ANOVA using Prism (GraphPad Software,Inc.). Result for UKE1 cells are shown in FIG. 2(A); and results forSET2 cells are shown in FIG. 2(B).

Example 3: Inhibition of Neoplastic Erythroid Colony Formation ofJAK2-V617F(+) MPN Hematopoietic Progenitors by Compound 1 andSynergistic Inhibition in Combination with Ruxolitinib at Very Low Dosesof PIMi

Peripheral blood mononuclear cells from an MPN patient (PV, JAK2-V617F+)were plated in methylcellulose containing cytokines withouterythropoietin (Epo), and in the presence of either DMSO-vehicle, 50 nMruxolitinib (Rux), 20 nM or 5 nM Compound 1 (PIMi) or the two drugs incombination. Epo-independent erythroid colonies were scored after 12days. Results are shown in FIG. 3. Expected % growth of combinationsusing the Bliss independence model is indicated by the transparent barswith dotted lines. The lower measured % growth indicates synergy.

Immunoblot analysis of MPN patient granulocytes left untreated (DMSOonly) or treated with the indicated concentrations of Compound 1 (PIMi)or ruxolitinib. Results are shown in FIG. 4.

These results indicate that Compound 1 and ruxolitinib have asynergistic effect at 1 nM, 5 nM, and 20 nM Compound 1 is unexpected.

Example 4: Conversion of Synergistic Effects to Human Doses

Plasma samples from patients were incubated ex vivo with the KMS12BMcell line for 2.5 hours at 37° C. Whole cell lysates were prepared fromthe cells, and levels of pBAD were determined using an ELISA. Datapresented are at 24 hours post dose (FIG. 5(A)) and in a time course(FIG. 5(B)), on cycle 1, day 8 for 80 mg BID of Compound 1 (averageinhibition of 76%). The results in FIG. 5(A) indicate that the IC₅₀ attrough for Compound 1 is reached at 65 mg BID. In turn, Compound 1 hasan IC₅₀ of 134 nM when BAD phosphorylation was measured in whole bloodsamples spiked with KMS-12-BM cells treated ex vivo with Compound 1.Hence, 65 mg BID of Compound 1 results in an IC₅₀ of 134 nM in thiswhole blood assay. As these results are in a whole blood assay unlikethe assay in Example 3, an adjustment must be made for protein bindingof Compound 1 in the whole blood assay.

Accordingly, the protein binding of Compound 1 was determined in humanserum (in vitro) ranging from 0.3 μM-10 μM. Compound 1 exhibited highprotein binding. For human, the average in vitro serum fraction unbound(fu) of 7.0%. For the protein binding determination, theMulti-Equilibrium Dialyzer System™ and diachema membranes from HarvardApparatus (Holliston, Mass.) were utilized. Samples were proteinprecipitated, matrix matched and then centrifuged. The supernatant wastransferred and analyzed using LC-MS/MS. The percent fraction unboundfor in vitro samples was calculated using final peak area ratios inpost-dialysis buffer divided by final peak area ratios in post-dialysisserum, with correction applied for volume shift.

The assay in Example 3 involved plating cells from blood in media. Dueto the protein content in the media, the free fraction of Compound 1 inmedia is 58%. The serum free fraction and media free fractions of 7% and58% can be used to convert the results in Example 3 to an human doseassuming a linear dose-response curve. This can be done by calculatingthe protein-binding adjusted activity as (cell activity*% free inmedia)/% free in serum—e.g., for cell activity of IC₅₀ of 1 nM, then (1nM*58%)/7%=8.3 nM—an 8.3-fold shift in activity.

TABLE 1 Cell activity - Protein-binding Example 3 adjusted activityHuman dose BID Daily human dose 1 nM 8.3 nM  4 mg BID  8 mg/day 5 nM 41nM 20 mg BID  40 mg/day 20 nM  166 nM 80 mg BID 160 mg/day

The 50 nM ruxolitinib in Example 3 corresponds a dose of ruxolinitb ofabout 15 mg BID. Generally, a dose of ruxolitinib in the 5-25 mg BIDrange would be expected to have synergistic effects.

Hence, the protein-binding adjusted activities of 41 nM and 8.3 nMcorrespond to IC₂₃ and IC_(5.7) for Compound 1. It was unexpected thatsynergy with ruxolitinib was observed in MPN cells at such low doses ofCompound 1—well below IC₅₀.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including withoutlimitation all patent, patent applications, and publications, cited inthe present application is incorporated herein by reference in itsentirety.

1.-30. (canceled)
 31. A method of inhibiting or ameliorating amyeloproliferative neoplasm in a human patient in need thereof,comprising administering to said human patient a dose from about 2mg/day to about 160 mg/day ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, and about 10 mg/day toabout 50 mg/day of ruxolitinib, or a pharmaceutically acceptable saltthereof.
 32. The method of claim 31, wherein the ruxolitinib, or apharmaceutically acceptable salt thereof, is ruxolitinib phosphate. 33.The method of claim 31, wherein theN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is selected from thephosphoric acid salt, dihydrochloric acid salt, hydrochloric acid salt,maleic acid salt, adipic acid salt, hydrobromic acid salt,(R)-(−)-mandelic acid salt, salicylic acid salt, benzoic acid salt,benzenesulfonic acid salt, L-pyroglutamic acid salt, methanesulfonicacid salt, (1S)-(+)-10-camphorsulfonic acid salt, fumaric acid salt,sulfuric acid salt, L-tartaric acid salt, and D-tartaric acid salt ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.34. The method of claim 31, wherein theN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is the phosphoric acidsalt ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.35. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 5 mg/day toabout 100 mg/day.
 36. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 80 mg/day.
 37. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 60 mg/day.
 38. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 50 mg/day.
 39. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 40 mg/day.
 40. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 8 mg/day toabout 20 mg/day.
 41. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 2 mg BID toabout 50 mg BID.
 42. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 40 mg BID.
 43. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 30 mg BID.
 44. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 25 mg BID.
 45. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 20 mg BID.
 46. The method of claim 31, wherein the dose ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is from about 4 mg BID toabout 10 mg BID.
 47. The method of claim 31, wherein the dose ofruxolitinib, or a pharmaceutically acceptable salt thereof, is about 10mg/day to about 30 mg/day.
 48. The method of claim 31, wherein the doseof ruxolitinib, or a pharmaceutically acceptable salt thereof, is about10 mg/day.
 49. The method of claim 31, wherein the dose of ruxolitinib,or a pharmaceutically acceptable salt thereof, is about 20 mg/day. 50.The method of claim 31, wherein the dose of ruxolitinib, or apharmaceutically acceptable salt thereof, is about 30 mg/day.
 51. Themethod of claim 31, wherein the dose of ruxolitinib, or apharmaceutically acceptable salt thereof, is about 5 mg BID to about 25mg BID.
 52. The method of claim 31, wherein the dose of ruxolitinib, ora pharmaceutically acceptable salt thereof, is about 5 mg BID to about15 mg BID.
 53. The method of claim 31, wherein the dose of ruxolitinib,or a pharmaceutically acceptable salt thereof, is about 5 mg BID. 54.The method of claim 31, wherein the dose of ruxolitinib, or apharmaceutically acceptable salt thereof, is about 10 mg BID.
 55. Themethod of claim 31, wherein the dose of ruxolitinib, or apharmaceutically acceptable salt thereof, is about 15 mg BID.
 56. Themethod of claim 31, wherein the myeloproliferative neoplasm is selectedfrom polycythemia vera (PV).
 57. The method of claim 31, wherein themyeloproliferative neoplasm is selected from essential thrombocythemia(ET).
 58. The method of claim 31, wherein the myeloproliferativeneoplasm is selected from primary myelofibrosis.
 59. The method of claim31, wherein the myeloproliferative neoplasm is selected from chronicmyelogenous leukemia (CML).
 60. The method of claim 31, wherein themyeloproliferative neoplasm is selected from chronic myelomonocyticleukemia (CMML).
 61. The method of claim 31, wherein themyeloproliferative neoplasm is selected from hypereosinophilic syndrome(HES).
 62. The method of claim 31, wherein the myeloproliferativeneoplasm is selected from systemic mast cell disease (SMCD).
 63. Themethod of claim 31, wherein the myeloproliferative neoplasm is selectedfrom chronic neutrophilic leukemia (CNL).
 64. The method of claim 31,wherein the myeloproliferative neoplasm is selected from chroniceosinophilic leukemia.
 65. The method of claim 31, wherein theruxolitinib, or pharmaceutically acceptable salt thereof, and theN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, are administered orally.66. The method of claim 31, wherein the ruxolitinib, or apharmaceutically acceptable salt thereof, is ruxolitinib phosphate andtheN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof, is the phosphoric acidsalt ofN-{(7R)-4-[(3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl]-7-hydroxy-6,7-dihydro-5H-cyclopenta[b]pyridin-3-yl}-6-(2,6-difluorophenyl)-5-fluoropyridine-2-carboxamide.